The mouse mutant lethal spotting (ls) encodes the vasoactive peptide endothelin 3 (EDN3) and the piebald (s) locus encodes its receptor, endothelin receptor B (EDNRB). Mice homozygous for mutations in either of these genes exhibit aganglionic megacolon and a white spotted coat due to the lack of neural-crest derived enteric ganglia and melanocytes in affected portions of the colon and skin. In order to understand the role of these genes in melanocyte development, we are using in situ hybridization to determine the time that these genes are expressed relative to other neural crest markers in mutant and normal embryos. A 1.3 kb cDNA clone of EDN3 was isolated from a mouse embryonic day 10.5 cDNA library and a 950 bp cDNA clone of EDNRB was isolated by RT pcr using gene specific pcr primers. These genes have been used to determine expression patterns in adult and embryonic tissues. We have shown that both genes are expressed very early during mouse embryonic development in neural crest derived cells and expression patterns are altered in various neural crest mutants. In order to determine the function of EDN 3, we are determining how exogenously added EDN3 alters the timing, number, survival and morphology of melanocytes in an in vitro system that we have developed. We have found that EDN3 can successfully replace TPA in this system and results in a 50 fold increase in melanocytes. We have found that of the related family members (EDN1 and EDN2) only EDN1 can substitute for EDN3 in this assay. We have also shown that the effect of EDN3 is dependent upon the presence of its receptor EDNRB on neural crest cells by using chemical inhibitors and piebald mice as donors for the neural crest cultures.